Document 99OD9nGqbL4n9n0KBEkOa9x77
.
SRPT 636
AR226-1527
Study No. T-6316.9; DT21
Fluorochemical (FC) Levels in Naive Rats
Final Report, Revision A
January 09, 2002
IInn--LLiiffee EStnadrtDDaattee::JJuullyy288,, 11999988
Study Location: 3M Strategic Toxicology Laboratory Corporate Toxicology 3M Medical Department
3M Center, Building 270-SB-314 St. Paul, MN 55144
RECEIVED OPPT NCIC
Study Director: Deanna J. Luebker, M.S., Advanced Research Toxicologist
3M Medical Dept. / Corporate Toxicology 3M Center, Building 220-2E-02 Saint Paul, MN 55144
Ph: 651-737-1374 FAX: 651-733-1773
2003 OCT-6 PM 12:27
Sponsor: 3M Corporate Toxicology
3M Center Building 220-2E-02
St. Paul, MN 55144
.
Research Client: 3M Specialty Chemicals Division 3M Center Building 236 St. Paul, MN 55144
}
TIF 1280
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DT21; T-6316.9 FinalStudyReportRevision A
01/09/02
Purpose of Revision: `The purpooftshies revision is to incoalrdatpaavaoilarbleatodtateeon the Naive Rat Study (T-6316.9) and the rat chow/fishmeal analysis. `This includes data reported in the initial final Naive Rat Study report written by Corporate Toxicology in 1998 (1), a summaryreporton the liver PROS levels written by 3M Environmental in 1998 (Appendix 1), and a summaryreportofthe liver PROS levels and rat chow/fishmeal analyses written by 3M Environmentialn 2001 (Appendix 3). Study Background: In an attempt todeterminethesourceof low-level PFOS body burden foundincontrol rats involved in some 3M contract dietary studies, a comprehensive plan with the following objectives and responsibilities was designed:
Ohbjeoctiuarv1eeas-oTfotihiencvnuesrtrigegnattedpioetteanrtyisatlusdoiuesrcoensopfercfolnutoarminiantaetditoenstwcitohmipnotuhnedsstuadty Covance Madison. Andrew Seacat was appointed study directorwithMarvCase as alternate. Jim Wolters, 3M Environmental, is responsible for coordinating and conducting direct air monitoring and wipe samples. OPbFjOeSctsieev2nei-nTcoondtertoleramtisneiniftchoenttwaom-iyneaatriodinoetfafryeesdtuidsyloefadNi-nEgtthoylthFeOlSowElevels of conducted at Covance Madison. Andrew Seacat was appointed study director with Marv Case as altemate.
Objecti3v-e To investigatethebackground serum and liver PFOS levels in naive
ratsofdifferent age groups from different sources. Information on the various diets supplied by the different vendors is to be obtained. Deanna (Nabbefeld) Luebker was appointed study director withMarvCaseas altemnate. `taOibnjteecdtfieve4de,-eTxopoisnuvreestiingartaettrhoeopmossosirbialcitytohatmPRbOSoifebxonptohsa.uMreatrisvsCitaesomemwinnagsfrom appointed study director. 3M Environmental Analytical Laboratory was deemed responsible for chemical analysis of samples gathered in objectives 1-4. `The study to examine objective 3 is complete and is the focus of this report. Once data addressing each objective are available, reports of the various studies will be generated.
Report Summary:
`The purpose of this study was to assess "endogenous" levels of fluorochemicals (FCs) in untreated Sprague Dawley rats of three different age groups acquired from three different breeders to determine what perfluorooctane sulfonate (PFOS) levels, if any, were present. Each vendor was contacted to provide information on the feed provided totheirrats while at their facilities. Levels of PFOS in the rodent feed supplied by the different vendors and in fishmeal were determined.
.
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DT21; T-6316.9Final StudyReportRevision A
01/09/02
`The liversofmale and female untreated SpragueDawley rats 6-8 weeks old, 10-14 weeks old, and approximately 9-12 months old (old retired breeders, ORB) from Charles River, Harlan,andTaconic Farms Laboratories were examined for iver PFOS levels. Male rat liversfromCharles Riverand Taconic Farms test animals showed significant endogenous levelsof PFOS that roughly correlated with age. Livers collected from the oldest group of `maleratsfromTaconic Farms contained significantly more PFOS than any other group in the study. The liversofthe female rats from Charles River and Taconic Farms contained very consistent levelsof PROS, showing no correlation with age. The livers of test `animals from Harlan did not contain PFOS above the limit of detection (1S ng/g). Based on these data, PFOS had bicaccumulated to a much great degree in male Charles River and Taconic Farms than in female rats from the same vendors. This difference may have been a result of PFOS being released in themilkand/ortransferred in uterotothe pups in females that had bore and nursed multiple litters. Female ratsmayalso more readily excrete PFOS through urinary excretion thanmalerats. Results of the vendor feed/diet history survey revealed that fishmeal wastheprimary ingredient in feed supplied to `Taconic Farms rats and the Sth listed ingredient in feed providedto Charles River rats. Fishmeal was not listed as an ingredient feed supplied to Harlan rats. Detectable levels of PFOS were found in samplesofthe chow fed to both Charles RiverandTaconic Farms rats. NoPFOS couldbedetected in chow provided to Harlan rats (fishmeal free). Analyses of fishmeal (six types arising from a minimum of 3 kinds of fish) suggest that it may be the source of PFOS in the chow. `To definitively make this conclusion, analysis of theactual fishmeal incorporated into each of the chow samples analyzed is required. Another possible source of PFOS is the paper coating used in feed bags. Further information on feed packaging may be gathered in a future study. Methods: `This study was performed in the 3M Strategic Toxicology Laboratory under adefined protocol (2). All in-life procedures were approved by the 3M Laboratory Animal Review Committee (3M LARC). `Thirty Sprague Dawley rats, fivemaleand five female fromeachofthe following age `groups: 6-8 weeks old, 10-14 weeks old, and ~ 9-12 weeks old (old retired breeder, ORB) `wereordered from Charles River, Harlan, and Taconic Farms. Each vendor was asked to provide the name of the feed provided to their rats, name and location of feed vendor, feed ingredient list and information on feed packaging. All animals remained in their shipping containers between arrival at 3Mandenthanization. No food or water, other than that provided in the shipping containers, was furnished. Within one hourofarrival at 3M, rats `were weighed, grossly examined, and euthanized by CO,asphyxiation. Sera and liver `were harvested and sent to Kris Hansen, 3M Environmental Laboratory - Fluorine Analytical Chemistry Team (FACT), for PFOS analysis. Liver specimens were analyzed as described in the Experimental Section of Appendix 1. `Sera samples have not been analyzed but will be retained for possible future analysis. Statistical analysis to detect significant differences (p<0.05) in liver PFOS concentration
Page30f23
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DT21; 6316.9Final StudyReportRev0i1s/0i9o/n0A2
between age groups, sexes, and vendors in the Charles RianvdTaeconric Farms rats was `performed using a one-tailed, homoscedastic T-test. The rate of change in PFOS levels (ppbiweek) was estimated by dividing the change in PFOS concentration by the number of weeks evaluated (approximately 38.5 weeks).
Rat chow from eachofthe vendors and fish meal (six types arising from a minimumof3 kindsoffish) were analyzed for POS concentrations as described in Appendix 2.
Results:
In life data including body weights, liver weights, and liver/body weight ratios as well as liver PFOS concentrations can be found in Tables 1-3. Detailed resultsofthe FC analysis, including graphs and a full data table, can be found in Appendix 1. Liver PFOS levels in CharlesRivermale and femalerats at 6-8 weeofkagse averaged 43.04.07 and 66.6 19.27 ppb, respectively. At 10-14 weeksofage, liver PFOS levels inmaleand female CharlesRiverratshadincreasetdo103.0 21.33and 79.9 12.06 ppb, respectively. Liver PROS levels in Charles River male and female ORBs were 145.0 +1242 and 67.8 + 29.63 ppb, respectively (Sec Table 1 and Appendix 1). LiverPFOS levienlalslHarlanratswerebelowthemethoddetectionlimitof 15ppb (See Table 2 and Appendix 1). Liver PFOS levels in Taconic Farms male and female rats at 6-8 weeksofage averaged 60.2 12.21 and 73.0% 12.29 ppb, respectively. At 10-14 weeks of age, liver PFOS levels in male and female Taconic Farms rats had increasedto92.2 +2275 and 75.7 + 24.40 ppb, respectively. Liver PFOS levels in Charles River male and female ORB were 327.0 115.37 and 65.6 + 27.24 ppb, respectively (See Tabl3e and Appendix 1). `The average rate of change in PFOS liver concentration for male and female Charles River rats from week 7 toweek45.5 was estimated to be approximately 2.6 and 0.031 ppb/week, respectively. The average rate of change in PROS liver concentration for male and female `Taconic Farms rats over the same time-period was estimated to be approximately 6.9 and-- 0.19 ppbiweek, respectively. Results of the T-test are included in Table 4. The change in liver PFOS concentration between age groups in male Charles River and Taconic Farms rats was significant at all timepoints (p <0.05). Livers from CharlesRivermale rats contained significantly more PFOSatall age groups than did livers of Charles River female ratsofthesameage group. Liversofmale Taconic Farms ORBs contained significantly more PFOS than livers of female Taconic Farms ORBS. Livers ofmale Taconic Farms 6-8 week and ORB rats contained significantly higher PFOS concentrations than did male Charles River rats of the same age groups.
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:
DT21; T-6316.9FinalStudyReportRevision A
01/09/02
VendorFeed/DietSurvey
ICnhtaerrlneastiRoinvaelrInrca.t,s BarreenftewdoPoMd,IMSOL.79PRMatIafnededMoisupsaeckDaigeetdsuipnpplaipeedrbbyagPsMIcoNautterdiwtiiotnh a
papercoating Intemational
material produced Inc., are as follows:
by
3M.
The
feed
ingredients,
as
listed
by
PMI
Nutrition
BgrHoAu,ndfiysehllmoewalc,oaml,fawlhfaeamteamli,dcdalniengmso,lassosyebse,ancamlecailu,macnairmbaolnaftate,prsaelste,rved with cyanocobalamin (source of vitamin B-12), biotin, DL methionine, calcium
pvainttaomtihnenAataec,etfaotlei,cdaic-iadl,prhiabotfolcaovpihne,rcyhlolaecceatlactief(esrooulr(cseooufrcveiotfamviintaEm),inthDi-a3m)i,n, `mmaegnnaedsioinuemdoixmiedteh,yslpordiimuimdisneloelnibties,ulnfiictoet(insiocuracceido,fpvyirtiadmoixninKe ahcytdirvoictyh)l,orsiidliec,on fdeirorxoiudse,ccarablocniautme,iozdiantce,sumlafnatgea,nzoiuncsooxxiiddee., copper sulfate, cobalt carbonate,
`HTaerkllaadn,rMatasdiarseonf,edWHIa.rlTaenkTleakdlfaededLMisa-l4s8o5pMacokuasgee/dRaitnpSatepreirlibzaagbsl.e DIitetissuunppklnioewdnbyatHtahrilsan
time, however, ingredients, as
whether ornotthese listed by Teklad, are
bagsarecoated as follows:
with
a
3Mmaterial.
The
Teklad
feed
sgoryobuenadncooiml,,csoomybgelauntemneamle,alg,rocaulncdiouamtsc,arwbhoenaattem,idddilcianlgcsi,umalpfhaolsfaphmaetael,, Db-raecwteirvsatderdieadnyiemaaslt,siteordoilz(esdosuarltc,eLo-flyvsiitnaem,iDnLD-)me,thviitoanmiinne,EvsiutpapmlienmAe-natc,etnaiatcei,n, c`maelncaiduimopnaentsootdhieunmatbei,surlifbiotfelcavoimnp,ltehxia(msionurmcoenoonfivtirtaatmei,npKy)r,idfooxliinceachiydd,robcihotlionr,ide, cvoiptapmeirnBsu,l;fatseu,ppzlinecmeonxti,dec,aclaclicuimucmaribodoantaet,e,combaanltgacanrobuosnaotxei.de, ferrous sulfate,
`TTacAoCn#i3c1.Faisrpmascrkaatgseadreinfead"TcoAmCme#r3c1iaRloldyeanctcDeipettasbul3peplpileydlbaymiZniaglteerd Cpaop,erGabradgn"e.r,ItPiAs. uinnkgnreodwiennatstitnhiTstAiCm#e3w1h,etahselriostrednboyt Zai3glMemraCtoe,riaarleaiss ufsoeldlionwst:hesebags.Thefeed
gfriosuhnmdea#l2, yseolylboewancommea,lg,raolufanldfawmheoalle,ocatosm, wghluetaetnmmiedadll,inggrso,uBnrdewwheorl'es wdrhieeadt,yeast, soybean oil, salt, dicalcium phosphate, ground limestone, vitamin and mineral remixes.
RoFed edPFe OSAn nalyt ses
Page Sof 23
Le
DT21; T-6316.9Final StudyReportRevision A
01/09/02
PMI Lab diet, fed to rats obtained from Charles River contained approximately 18 ng/g PFOS. Rodent dietfrom Zigler, fedtorats purchasedfromTaconic Farms contained approximately 12 ng/gof PFOS. Tekladrat/rodentdiet, fed toratsobtained from Harlan (fishmeal free), didnotcontain PFOSabovethe limit of quantitation (LOQ) (see Appendix 2).
`FishMeal PFOSAnalyses
Six types of fishmeal arising from at leastthreetypes of fishwereanalyzed for PFOS (see Appendix 2). PFOS was detected in 50%ofthe fishmeal samples analyzed. Itis not known, however, what type of fishmeal was incorporated into the various rat chows. Therefore,itcannotbedefinitively concluded that fishmeal is the source of PFOS in the rat chow. Analysis of the actual fishmeal incorporated into the chow is required to provide a direct link.
Conclusions: Basedonthesedata, PFOShadbioacctouammucuh glreaatdtegreeedinmalerats from CharlesRiverandTaconic Farmsthaninfemaleratsfromthesesamevendors. This differencemayhavebeen a resultof PFOSbeingreleasedinthemilkand/ortransferriedn utero to the pups in females that had bore and nursed multiple litters. Female rats may also morereadilyexcrPeFtOeSthroughurinaryexcretionthanmalerats.Theliversoftest animals from HarlandidnotcontainPFOS above thelimitofdetecti(o1n5 ng/g). Results of the feed analyses revealed detectable levels of PFOS in samples of the chow fed to both Charles River and Taconic Farms rats. No PFOS couldbedetected in chow provided to Harlan rats (fishmeal free). Analyses of fishmeal (six types arising from a minimum of3 kinds of fish) suggest that it may be the source of PFOS in the chow. To definitively `make this conclusion, analysis of the actual fishmeal incorporated into each of the chow samples analyzed is required. Another possible sourceof PFOS isthepaper coating used in feed bags. As stated in the results section, 3M suppliespapercoating to PMI, the `manufacturer of Charles River PMI chow. It is not known, however, if Harlan and Taconic Farms feed suppliers also use 3M material in their bags. Further information on feed packaging may be gathered in a future study.
Page 60f 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
List of Tables:
1) Charles River In-Life and Liver PFOS Data 2) Harlan In-Life and Liver PFOS Data 3) Taconic Farms In-Life and Liver PFOS Data 4) T-Test Results (p values) - Charles River and Taconic Farms Age, Sex, and Vendor
Liver PFOS Comparision.
Page 70123
DT21; T-6316.9 Final Study Report Revision A 01/09/02
[Body wt(g)
iver wt/ [PFOS Liver Conc. (ng/g or ppb), [bodywt [individual _|ave [stdDev
craresT 1 1 TTTTT T |
river R1 [c8wks | 171.7] 830] 0.04
"3085 43.0]
R2 o8wks | 179.6] 830 0.04
488)
R3 o8wks | 161.5) 7.05] 0.044
426)
R4 le-8wks | 151.6] 6.60 0.04
39.5]
RS o-8wks | 166.8) 8.08] 0.048
411
R6 [F[6owks | 173.7] 8.69] 0.050]
549] 66.6
R7 [F [e8wks | 1683 7.13) 0.042]
62.)
Re [F [6-8wks [165.1] 7.46 0.045]
65.4
Ro [F [68wks | 1417] 491] 0.033]
99.6]
RIOJF _[6-8wks | 1507] 647] 0.041
51.2)
RIM [10-14wks| 524.3] 10.55 0.039)
100.0}
R12M [10-14wks| 367.9 12.66) 0.034]
140
RIBIM [10-14 wks| 356.7] 13.10 0.037]
96.8)
Ri4IM [10-14 wks| 358.5] 13.23 0.037]
878
R15[M [10-14 wks| 365.6] 13.65] 0.037]
904
[CRi6[F [10-14 wks| 226.1] 10.49] 0.046]
722] 799)
lor17[F [10-14wks| 199.5 6.32] 0.032]
939)
lcR18[F [10-14 wks| 203.4 6.13 0.030]
71.4]
ICR1s[F [10-14wks| 196.4f 7.14 0.036]
922)
jcR2o[F [10-14 wks| 209.8] 7.08] 0.034]
696
[CRzilM `ORB | 4062] 9.75] 0.024]
131.0]
2
cream |orRB 3423 10.09 0.029)
133.0)
lcRza[m [ore 395.1 10.83 0.027]
151.0)
|cR24[m [oRB 3637| 9.24 0.025
149.0)
|cRzs[m__|ore 460.2] 1393 0.030)
160.0}
[CRzs[F [ore 3942 12.70 0.032]
1190] 67.9)
lcrz7[F [ore 4125) 16.09 0.039)
67.
lcR2sF [ORB 4829) 14.80 0.031
45.0
lcReo[F [ore 485.3) 14.96 0.031
55.5]
lcRsolr_jore 4242] 1134] 0.027]
53.0)
"ORB = oid retired breeders
[* Outlier- not used in calculations.
[Method detection limit (MDL): PFOS = 15ng/g or ppb
Page 8.0f23
:
DT21; T-6316.9FinalStudy ReportRevision A
01/09/02
[AgeGrp. [Body [Liver wt(0) [wt (g)
PFOS LiverConc. (ng/gorppb) individual _|Ave [stdDev
1 T_TTT T
AT
[68wks | 198.4] 9.45 0.048]
IH2
6-8wks | 201.7] 9.34] 0.046]
[Ha 6-8wks | 2034) 850] 0.042]
[Ha
6-8wks | 2039) 838 0.041
IHs
6-8wks | 204.2] 9.17] 0.045)
[He |F_[c8wks | 2143 7.62 0.03]
7 |F [68wks | 2080] 7.19] 0.005]
He |F [68wks | 2128) 7.4] 0.004]
Ho |F [68wks | 2003) 7.30] 0.05]
H10 JF [6-8wks | 2090] 8.08] 0.039)
[FT 10-14 wks| 300.6] 10.69] 0.036)
[H12 10-14wks| 307.9f 12.33 0.040]
[H13 10-14wks| 304.0f 11.28) 0.037]
[H14 10-14 wks| 209.6] 10.09) 0.034
[H15 10-14wks| 306.0f 11.38] 0.037]
[H16 [F [10-14wks| 237.7] 6.65] 0.028)
H17 [F [10-14 wks| 241.1] 7.20] 0.030]
H18 |F [10-14 wks| 230.7] 7.14] 0.00)
H19 |F [10-14 wks| 236.3] 8.54) 0.036]
20 |F [10-14 wks| 2334] 7.33) 0.031
[FT [FORE | 470.7] 17.00] 0.036]
[H22 ors. 4628] 14.50 0.032)
IH23 ORB 446.6] 15.55 0.035
[Hoa lore 4582] 1452 0.032]
[Hes oRB. 4789] 11.19] 0.023
[Fs [F_ [oR 337.8] 10.45] 0.031
He7 [F [ors 3262| 1027] 0.031
Hes [F [ore 2857) 854 0.030)
Heo [F |ORB 2862| 9.01] 0.031)
Hao [F [ors 306.9] 11.00] 0.036]
1 1|
MDL| <MDL| <MDL] <ML| <MoL| <MDL] MDL] <MDL] <MDL <ML| <ML| <MDL| <MDL| <MDL| <MDL| <MoL| <MDL| <MDL| <MDL| _<MDL| <MDL| <MDL| <MDL| <MDL| MDL] <MDL| <MDL| <MDL| <MDL| <MDL| MDL] <MDI <MDL| <MDL|
<MDL{"
<MDL]
|* ORB = old retired breeders [Method detection limit (MDL): PFOS = 15ng/g or ppb
Page 9.0f23
DT21; T-6316.9 FinalStudyReport Revision A
01/09/02
meme
[FARMS
[Body [Liver
PFOS Liver Conc. (ng/g or ppb)
[ 1 T1 1 TT | wt(9) |wt (9)
individual |Ave
[Std Dev.
F1
[6-8 wks 2002] 9.64] 0.048
63.6}
12.21)
F2
16-8 wks 202.4] 10.18] 0.050)
63.0}
F3
16-8 wks 184.1] 9.28) 0.050)
47.7)
F4
16-8 wks 187.5 10.26 0.055
49.2
F5
16-8 wks 1836 9.34] 0.051
77.5|
F6 |F [6-8 wks 201.2] 9.73] 0.048
67.0|
9)
F7 [F [6-8 wks 189.9 8.40 0.044
89.3
8 |F [6-8 wks 1725) 9.40] 0.055
78.8]
9 |F [6-8 wks 207.9) 10.28) 0.049
73.2
F10|F [6-8 wks 199.5| 10.54] 0.05
56.6]
F11
[10-14 wks| 454.3] 17.97] 0.040|
86.5]
22.7
F12
10-14 wks| 334.6] 13.07| 0.039
54.7]
F13
10-14 wks| 459.9] 17.65 0.038]
F14
10-14 wks| 407.5 14.1 0.035
F15
10-14 wks| 317.8] 12.75] 0.040}
84.9] 119.0| 86.2]
F16
10-14 wks| 214.4] 9.
0.044}
52.9) 75.7|
0
P17
10-14 wks| 226.1] 8.94 0.040)
71.5)
F18
10-14 wks| 216.1] 8.4 0.039
72.6
F19
10-14 wks| 241.0) 11.21 0.047|
64.4]
F20
[10-14 wks| 246.5] 8.5 0.035
117.0)
F21
"ORB
F22 JORB
F23 JORB
687.8] 561.2| 456.0)
22.22] 18.24] 14.96]
0.032) 0.033] 0.0:
151.0] 441.0] 379.0]
327.0] 115.3:
F24 F25
JORB JORB
525.7) 17.91) 586.6] 18.24]
0.034 0.031
390.0] 276.0
F26 |[F
F27|F F28|F
[ORB
|ORB |ORB
281.5]
281.4] 247.5)
19.56]
18.81) 15.65
0.069]
0.067] 0.063]
59.5 65.6]
4
105.0)
71.9]
F29 |F |ORB
289.5) 14.95 0.052
63.0]
F30 [F |ORB.
338.7| 17.58] 0.052]
29.0]
|" ORB = old retired breeders [Method detection limit (MDL): PFOS = 15ng/g or ppb.
Page 10 0f 23
DI21; T-6316.9 Final Study Report Revision A 01/09/02
able 4: T-Test Results (p values) - Charles River and Taconic Farms Age, Sex, and Vendor
Liver PFOS Comparision
[Vendor|
`Age/Sex Group Comparision
[658 [10.14wk [6BWK& [68& [10-14wk [6Bwk& [6-8 wk [10-14wk [ORB 10-12wkla ORB |oRB |10-14wk |s ORB |oRB 8 6-8 wk |& 10-14wk [ORE
charies
onl 021] 04
00
River
Taconic} Farms
|
oz8l 030] 007 Vendor Comparision
025 0.00
[male[Fem __Ta ooocoml somoome oom |
Chartes [68 wx [10-1awk [ORB& [6:8 wk [10-14wk |ORB&
River |s 68wk|& 10-1awi{oRE |s 68wk |aaw10k- lore
vs
Taconic}
0.13
0.37
Farms
TEST - one-tailed distribution, homoscedastic
Page 110123
%
:
DY21; T-63169FinalStayReportRevistas A OOO
Ip,TosicobopyServicesReport foxSadyNo. 63169;DT21.
Pe(s FC)Levelt fnNatvoo ras.Sepn t,1998.
2)3M MedicalDepartment, ToxicologyServicesProtocolforStudyNo. T-6316.9;DT21. Piuor (FCo ) Lec velsh inNe aivm erai ts.Jc ulya ,19l 98.
Page12023 .
-
Signatures:
Prepared By:
Deanna J. "a Ms
Study Director
Reviewed By: 2 2TZ
John Butenhoff, PhD., DABT Corporate Toxicology Management
(Inika Boned
AndrewSeacat, PhD,, DABT
Senior Research Toxicologist
DT21; T-6316.9Final StudyReportRevision A 01/09/02
Date
aud /, 2003 Date
(/9/2%~
Date
Page 13023
00902 :
DT21; T-6316.9FinalStudyReportRevision A
Appendi1x
3M Environmental Laboratory: Fluorine Analytical Chemistry Team
Contact:KisHansen
FluorineAnalyticalChemistry Team
so Building2-3E-09
Studyof PFOS levelsinNaive Rats
`Summary report
Experimenta Summary
ifferent sInuoprldecrstwoearsseesQs"enudogeanouasnn"alleyvtzeeldsfaoorfPPRRlOOSS.inTtherseteadniismtailnsc,ttahegelisvoefrsroatfs nwienertryreaptrsfersoemnttiehdrnee
thegroupof animalsreceivedfrom eachsupplier:6-8weeksold, 10-14weeks old,andretiredbreeders (>
14weeks). Thetestanimals,receivedbythe ToxicologyDepartmentat 3M, were sacrificeduponreceipt;
tissuesamplesweredelivered tothe3MEnvironmentalLabforextractionandanalysisby FACT.
[P Cle River |RR aigh:NC [PPuna [L10L10A[Rdo [11] Supplier
Location
wks
`male:female
TaconicFarms |Gernantows,NY_[TAC HT|__| 10 | do| ti]
*3Mis/was asupplierofpapercoatingmaterialtoPurina Mills, Inc. Curritiesnoatktnolwniyf3,M
suppliesmaterialtoTEKIador Taconic Farms.
AnalytiLciavleSrusmammaplreyswere homogenizedandextractedusinganion-paringreagent.Theextractswere
`analyzed quantitatively using high-pressure liquid chromatography-electrospray tandem mass spectrometry
r( eportH .ThP epreL sencC aenodre- avbalsueE antceedoS vfeortsM huesraknS neoxwtnM rafcltueS odrcoucr) hveem.icAanlaclyotnitcaamlidneatnatilssaanrdemaevtaaibloalblietienswtahesfull
`ascebrytinaspiecntieond.
Results Summary RatliversfromCharlesRiverand TaconicFarmstest animalsshowedsignificantendogenous
levofePFlOSs. Liverscollectedfromoldestgroupofmalerats from Taconic Farmscontainedsignificantly
`morePFOS thananyothergroupinthe study.
"Thelivers of testanimalsfromHarlandidnotcontainPFOSabovethelimitof detection(15ppb). PFOSlevelsintheliversofmaleratsfromCharlesRiverand TaconicFarmsroughlycorrelated
{`whietlhathsetaPgReOoSf,twhheialneimtahelsa. ,Thraettiirse,dlbiveerdscsolmleaclteedrfartocmotnhtaeiynoeudtngeehsitgmhaelsetractosn,ce6n-t8rwateieoknsold,contained
"Thelivers ofthefemaleratsfromCharlesRiverand Taconic Farmsweredeterminedtocontain
`vaerymcontsiiNsynottelhnziteleesvkdedlnyso.owfnPRlOuSce,oschhoewniincganloscomoarmirseanlswaoirttmhiaaogpenl.es wer deified nthe lve samples
Graphicalresultsand afulltableofresultsareattached.
Page140123
>
DT21; T-6316.9 Final StudyReportRevision A
01/09/02
`Currently,methodsarebeingdevelopedfortheaAnaplpyseisnodfil1xowlevels ofPFOSandethyl-FOSEalcoholin
samplesofchowfromeachsupplier.
`SEaxmpperliempentral eparasamptlesi,HPoLCn-ES-MSa:loqn-puaireingeoxtruactsion `Analyte isextractedfrom asamplematrixwithanion-pairingreagent (tetrabutylammonium
fhlyudorroogcehnemsiuclaflast.e (OTnBcAe))thienaanipHo-nc-oTnBtArplaleidriesnvfiorronmmeedn,tt.heaTnhaelyctaetiiosnitcrarnesafgeerraetdsienletcotiavneloyn-tparogleatrsorangiaonniicc:: solvent (ethylacetate),dried,andreconstitutedinmethanolforMSanalysis.
HPILnHCPaLnC-d,HPaELnCa-SlEiqSuMMoStoMSfS:eFxotrradcettiasileidnqjueacltietdaatinvedwoparsksedthrough areverse-phaseliquid cthortohmealtioqguriadpmhoibcicloelpuhmans.e,Btahseedaonanlyttheeiafsfrientiatiynoefdtfhoeranaalchyatreafcotretrhiestsitcaatmioonuanrtyopfhatsiemien.tFheocroelxuamnmrepllaiteniva,e: standardsolutionPFOSmay eluteat 10.5 minutes.Retentiontimesbetween astandardPFOSsolutionand theanalyteextractedfromgroundwateri thisanalysiswerematchedtowithin 19%on theHPLCsystem.
FollowingHPLCseparation,ESMSprovides arapidandaccuratemeansforanalyzing a wide rangeoforganic.compounds,including fluorochemicals. Elecatnironiozatsionptecrhniaqueyus,ed primarilyforthedetection ofmolecular ions,isgenerallyoperatedatrelativelymildtemperatures MoleculeEsSarMeSiMonSizaedd,dpsoassniabdldyiftriaognmaelndtiemde,nasnidodneotfeccteerdta.intytocompoundidentification.Asin ESMS,a `charaprcimtareyironissseltecitecd. However,insteadofsimplymonitoringtheprimaryion,inESMSMS theionisbombwitahhirgh-edneregygdas.As aresultofhigh-energycollisions,smallersecondaryionic fragmentsunique totheprimaryionarecreatedanddetected. "ThisioniFofrreaxgammepnltee,dfionrtPooRtOhSer(iCo1nFs;ssuScO5h)aasna8l0ysaims,uio(nco4r9re9siposnedliencgtteodaSsOtxh)e,c9ha9raamctuer(icsotricrepsrpiomnadriynigotno. F505), 130amu (correspondingto CF;SO5),180amu (C;F.SO5), and 230amu (CyFSOy). Eachofthese: secondaryfragmentsisdetectedandcanbe sedtodifferentiatePROSfromothercompoundsthatmight havethesamecharacteristic 499amuprimaryionbutdifferentchemicalcompositionsandsecondaryion fragmentationpatterns.
HPLCsysCtoelmu:mnH: ewleSetrietKs1-e100PyLiBqaesutiadcstChCkr1oo8macantoolrguermadnph
2X 100mm, umpartiscilze
FSloolwvernateA::
320.00mMmianmmonium acetate:
,
SoSlolvveeanttBG:radient: 4Me0t%hatnoo9l0% B in 8.5 minutes.
Holdat 90% Bfor 3minutes
HRoeltduamtt4004%0%BBfiorn|1mmiinuntuet:e
InRjuencttiimoen:volume: 10 uL. 13.5 minutes
ElecTtandremMoasssSppecrtroametyer MicromassQuattroII APImass specirometermassspectrometer
MassLynx3.1software
Page 150f23
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T-6316.9
Final
StadyReportRevision A ous
Conevoltage:
3060V Appendix1
Collgiasscioeorgny: ~~ 40eV.
Mode:
electrospraynegative
Sourceblocktemperat1u1r5eC;
DPersiomlavraytiloonn:temperature: 249590C
Daughter ons:
80,99,130, 180
Electrode:
Zspray
QualitycAolnltarnoallsysuemsmwearreyconduciedwith amoderatetohighlevelofqualitycontrol.Duplicatematrix spikeanalyseswereconductedforoneanimal fromeachgroupof animals.Exceptasnotedin theresults table,recoAvcearliiebsrwaetrioenwcihtehcinktsthaenadcacredptwaabslaernaalnygzeeodfe8v0e-r1y250-%1.0samplestomonitor instrumentaldrift. `Quantation wasbasedonlinearregressionanalysisoftwocurvesbracketingeachgroupofsamples. `QuantitationofPROS wasbasedontheresponseof3-4daughterionsoftheprimaryion.
Results Seespreaattadchesdtohtheisreepotrt.
FACTmembersparticipating: KHansen L.Clemen HM..EJloihenfssoonn G.Langenburg R.Wyme SL.AH.eSimmidtahh!
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DT21; T-6316.9FinalStudyReportRevision A 01/09/02
Appendi2x
SUMMARY OF NAIVE RAT/RAT CHOW/FISH MEAL STUDY ANALYSES
Conducted by the 3M Environmental Lab (prepared 03/01/01)
NaiveRats (aTnaikmaelnsf,rtohemlisvuermsmoafrnyirneeptoyrrtatissfsureodimtnh1r9e9e8d.i)ffIenroenrtdseurptpolaisesresswser"eeqnudaongteintaotuisv"elleyvaenlasloyfzePdFOfoSriPnFOtSes.t Threedistinctagesofrats wererepresentedin the groupofanimalsreceived from each supplier: 6-8weeks old, 10-14weeksold,andretiredbreeders(> 14weeks). RaPRtOlSi.veLrisvferrosmcCohllaerclteesdRfirvoemroalnddesTtagcroonuipcoFfarmmaslteersattasfnirmoamlsTsahcoowniecdsFiagrnmisfciocannttaeinndeodgsiegnnoiufsilceanvtellysmoofre. 'PFOS thananyothergroupin thestudy. "The liversoftestanimalsfromHarlandidnotcontainPROSabovethelitofdetection (15ng/g). PROSlevelsinthelivers ofmaleratsfromCharlesRiver and TaconicFarmsroughlycorrelatedwiththeage: oftheanimals. Thatis, liverscollectedfromtheyoungestmalerats,6-8weeksold,containedtheleast "PROS,whiletheold,retiredbreedermale atscontainedthehighestconcentration. "TcohnesliisvteernstolftehevfoefemPaFllOerSs,assfrhomCohnaorwlcoeirsrReinlvaetrigaonnwdiTtahcaognei.cFarmsweredeterminedtocontainvery
LimofiDtatsa:
or`iSgcrieneanlisngufmomraorthyreerkponrotwdnomcuemetntastabteodoftEhltat-iF"ONtSoEoe-tOhseHrk(en.og.wPnFflOuSoAr,ocPhReOmiScAaAlc)ownatasmpienrafnotrsmoerd.meTthaebolites i`nwetrheeidleinvteirsfiaemdpilnetsh,etlhievearnaslaymtpiclaelsLanOaQlymzaeydinontthhiasvsetbuedye.n" sHufofwieciveenrtt,boasseeedeoxnpetchteeldevmeetlasobofliPtFeOleSvoeblss.erved (Basedonthe 2yearsEt-FOSE-OHfeedingstudy,PROSAAlevelswouldbeexpectedt0beapproximately 9%ofPFOSlevels; POSAconcentrationswouldbe expectedtobeapproximately 5%ofPFOSlevels. reBlaactkicvaellcyhuliagthe-dd,otsheee2xypeeacrtfeedecdoinncgesnttruadtyimoansyoofrtmheaymentoatboplriotveisdaeraennaecacretphteab1l9e9m8oLdOeQlo.fDtahtisaflrowo-mletvheel exposure.) SpikerecoverystudieswereconductedforPROSin atliversandwereacceptable. Matrixspikestudies `conductedforotheranalyteswere, forthe mostpar,acceptable.
RatChow Ratchowfrom 4separatevendorswasinvestigated.Twoofthesourcesof atchowweredeterminedto containPROSabovethelimitofquantitation. NIH/PMILabdiet,fedtoratsobtainedfromCharlesRiverwasdeterminedtocontainapprox 18ng/g P`RaOSp.pSrmaollax1n2iimnmagl/adgioteftfePrFloOmySZ.eiTgleekri,afderdattioroadtesnptudriceth,asfeeddtforroamtsToabctoaniinecdFfarromsmwHaarsldaent,edrimdinnoetdtcoonctoanitnain PFOSabothevLOeQ. Liomftihe tDatsa:
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DT21; T-6316.9Final StudyReportRevision A
Appendi2x
01/09/02
`Spikerecoveriesstudieswereconductedoneachtypeofchowtestedand allresultswereacceptableinall
cases(77-98%). TheLOQforPFOSinratchowisapproxim2a-1t0engl/gy.
Fish Meal
`There isgreatvariabilityinboththesourceandtypesoffishmealavailablecommercially.Thisvariability complicatestheanalysisof fishmealandtheinterpretationofdataconcerningfishmeal.
Fishmealsusedforanimalandfishdietare typically generatedfromwhatare referredtoas "industrialfish". Industrial fish, including menhadenandherring, areusually quitehighinoil content andthus notfit for `human consumption. Approximately90%oftheworld's fishmealisproducedfromindustrialfish. Fishmealisprocessedinfactories ordirectlyon thefishingvesseland resultsintheproductionofseveral
`separatefishmealproducts. Afterthe fisharecooked, aliquor(il,water, andprotein)isseparatedfromthe `solidsby"pressing". Thesolidthatremainsafterpressingiscalledthe "presscake",theliquidcomponent, oilandsolubleprotein,is"stickwater". Subsequenttopressing,theoilisseparatedfromtheremaining `componentsofthe stickwater bycentrifugation. Afterfurther distillation,thisfishoilmaybedirectly incorpintooarniamatl ediedt.Theremainingstickwaterisevaporateddownto athick syrupof 30-50%
`solidsandmaybesoldas "condensedfishsolubles". Thesyrupmayalsobeaddedbacktothepresscake anddriedandsoldas "wholemeal".Presscakewithoutthesyrupis soldas "presscakemeal." Aol fthl ese `componentsmaybeincorpino toarniamatl feeedd.
`AccordingtotheUniversity ofFlorida CorporateExtensionService,goodqualityfishmealaverages60-
70% protein; the oil content ranges from 2-15%.
Fishmealisincorporatedinto foodfor avarietyofanimalsandfishincludingfarm-raisedsalmon,poultry, andcows(particduailraycrowls)y.
Inthis study,six typesoffishmeal,arisingfromatleast 3typesof fish,wereanalyzed.
Menhaden travelinlargeschoolsand migrate upthecoast eachyear. Theyarefoundinwarmer,near
shorewatersandintheUS,are fishedprimarilyoffAtlantic Coast. Menhadenconstitute 98%ofthe fishmealproducedinthe US.
' Capelin,also aschoolingfish,spendmuchoftheirlivesoffshoreinthedeepwatersofthe Northern
Pacificandthe Arcticandmovein-shoreonlytospawn.
The habitatfordifferentclassesofherringismuchmorefarrangingthanthatofeithermenhadenor
apelin. Differenttypesofherringcanbefoundfromthe NorthernPacificwatersoffthecoast of Canadaanddownto thesoutheAatlsantticecorasnt.Likemenhadenandcapelin, herring aresmall, `schoolingfishandthusare targetedforlarge-scalenetting.
`ProteinConcentrate (type offishmealnot specified) |( <LaOQppr3nog/gx ) |
Omega Protein FAQ (type offishnomtse pecaifiled
[Mesb~s~donF[i6s1m0eat|
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DT21; T-6316.9FinalStudyReportRev0i1s/i09o/n0A2
Appendi2x
Matrixspikestudies wereconductedon asingletypeoffishmeal (menhaden). PFOSspiiknteothde `menhadenfishmealwasrecoveredatapproximat6e6l%y. Matrixspikestudieswerenotconductedforthe remtyapesiof fnishimealntesgted. `The LOQ forPROS determinationinthsefishmealsampleswasapproximately3.5 ng/g.
Sc ummo arym of tp he3oneon fthit sstus dy:
Ratswith RatChowusedby |Rat Chowusedby Vendor VenodfRoatrs_| endogenous PFOS?| Vendor Contains PFOS?| Containsfishmeal?
Fe] ww
re
.
Becauseitis notknownwhattypeoffishmealwasincorporatedintothevariousratchow,itcamnotbe definitivelyconcludedthatfishmeal ithesourceofPROSintheratchow.Althoughindirectevidence indicatesthisprobability,becausePROSwasonlydetectedin 50% ofthefishmealsamplesanalyzed, `analysis oftheactualfishmealincorporatedintothechowisrequiredtoprovide adirectlnk. `Other variablesaffectingtheraspriortoshipmentto 3M (suchasPROSlevelsinwaterandinother `componentsofthefeed)havenotbeentested.Tothebestof mymemory,this chowhasnotbeentestedfor the potentialof EFOSE-OH transferfrompackaging. `Shouldfurtherworkbeconductedonthetissuesamples, a lowerlevelscreen formetabolitesandother knownfluorochemicalsi recommended. Thisinformationwouldprovideadditionalevidenceconcerning theoriginofPFOSidentifiedinratlivers. `The motivationtopursueanyof theseadditionalinvestigationsdependsonthecorporation'senduseofthis data. Theorisgtudiyobnjecativle: ...todeterminewhatPFOSlevels,ifany,arepresentinSprague Dawleyratsofthreedifferentagesfromthreedifferentsuppliers"(MedicalDep.protocolfo study # T6319.9)wascompleteduponissuanceoftheEnvironmentalLab summaryreportin 1998.
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